Diagnostic tests upon liquid samples and diagnostic tests utilizing liquids are in widespread use in medical technology, environmental monitoring devices, and commercial applications. A significant impediment to the utilization of many diagnostic testing processes has been the impractical delay required for chemical reactions in such processes to proceed to a meaningful completion. It is not uncommon for diagnostic chemical reactions occurring in a liquid system to proceed for extended periods of time, e.g., in excess of thirty minutes. Such delay may make certain diagnostic tests entirely unsuitable for situations in which timely results are needed.
In an emergency room, delay in obtaining results from a diagnostic test may delay accurate evaluation of a patient's condition, to the extreme detriment of the patient. Even under less critical circumstances, such as a routine visit to a doctor's office, an hour delay in obtaining results from a diagnostic test may hinder a doctor's diagnosis and treatment of a patient during a single consultation. Any delay in treatment could result in harm to the patient. At the least, an extended delay in obtaining test results may necessitate an additional follow-up consultation and office visit, thereby increasing the overall cost of treatment to the patient. In the laboratory, the slow chemical reaction time of a diagnostic test may significantly reduce the efficiency of research efforts and burden researchers. Further, time-consuming diagnostic testing in industrial chemical processes may dramatically increase manufacturing costs and reduce production volume.
To avoid the above-described consequences, apparatus and methodology for increasing the speed of diagnostic testing processes are greatly desired, especially in connection with assays that incorporate a binding reaction, e.g., immunoassays, nucleic acid hybridization assays, and receptor-ligand binding assays. It would be particularly useful to increase the reaction rate in assays utilizing binding reactions that involve the binding of components of a solution to reagents located at a solid-phase support. Such assays should provide precise, quantitative results and be highly sensitive. In addition, it is also desirable that apparatus for conducting diagnostic test assays be small, portable, low cost, robust, and easy to operate. The above considerations are especially important in the field of Point-of-Care (POC) medical diagnostic testing (e.g., testing done at home, at a hospital bedside, in an emergency room, or in a doctor's office).
It is believed that the rate of a binding reaction depends upon the mass transport rate of the reagents involved. For binding reactions that occur at a solid-phase support, the rate at which molecules in solution bind to reagents located at a solid-phase surface may be limited by the rate of mass transport of the molecules to the surface. When such systems are not subject to active mixing, molecules in solution reach the solid-phase surface primarily by diffusion through the solution. It has been found that diffusion rates are generally too slow to allow binding reactions to approach completion with a 30 minute period. In addition, the presence of small convection currents in the solution, e.g., due to temperature gradients, can cause the rates of a binding reaction to vary considerably and thus be difficult to predict and control.
There have been numerous prior attempts to improve the mass transport of molecules to a solid phase support in a binding reaction system. Considerable efforts have been directed to increasing mass transport rates through the introduction of controlled convection currents, e.g., by vortexing, by using stirring devices, or by passing a solution over a solid-phase surface in a flow cell arrangement. Such approaches commonly utilize relatively expensive and complex mechanical devices, such as solution stirrers or pumps, and, consequently, are not suited for use in an assay device that is small in size, robust, inexpensive to manufacture, and easy to use.
Also, a liquid ultrasonication bath to promote mixing has been described in U.S. Pat. No. 4,575,485 (Sizto et al.). Sizto et al. mention a container, holding a volume of assay medium and a “dip-stick” immersed in the medium, submersed in the bath of a conventional liquid-bath ultrasonic cleaning device. Ultrasonic vibrations from the shell of the cleaner bath are liquid-coupled to the container. The vibrations traveling through the liquid of the cleaner bath dissipate in the volume of the bath and reflect off of the container material and off of the shell of the bath. Such liquid-coupling is clearly inefficient and can dissipate considerable amounts of ultra sonic energy.
The exact nature of the ultrasonic vibrations being transmitted to the assay medium and to the dip-stick will significantly depend upon apparatus design and usage conditions. For example, the shape of the container for the assay medium, the shape of the shell of the bath, the position of the container in the bath, the position of the dip-stick in the container, the position of the source of vibrations, the amount of dissolved gas in the liquid in the bath, and the volume of liquid in the bath will each affect the transmission of ultrasonic vibrations. In use, the volume of liquid could easily change due to evaporation, splashing or release of gasses dissolved in the liquid in the bath. All of these may affect the vibration transmission characteristics of the bath.
Since small variations in structure and operational conditions will considerably affect the transmission of ultrasonic energy in a device according to Sizto et al., it can be expected that precise reproduction of particular ultrasonic bath conditions throughout the duration of a particular reaction will be extremely difficult, if not impossible, to achieve. Consequently, it will be extremely difficult, if not impossible to achieve reproducible assay results with such a device. Time-consuming chemical reactions sensitive to ultrasonic energy may not be reproducible at all. In addition, the use by Sizto et al. of a liquid bath ultrasonic cleaner device presents an unnecessary risk of cross-contamination between the bath and the assay medium. Such contamination is likely to cause erroneous assay results.
Further, an apparatus according to Sizto et al. is not particularly suited to commercial application. As a consequence of designedly incorporating a liquid bath, the apparatus of Sizto et al. is relatively large, cumbersome and heavy and consumes considerable electrical power. Such power is required because of the wasteful dissipation of ultrasonic energy in the bath shell, bath liquid, and assay container. Clearly, a device according to Sizto et al. very inefficiently transmits ultrasonic energy to an assay medium in a container and from there to a binding surface. Moreover, the use of an ultrasonication bath is an additional complicated assay step requiring skillful manipulation by a user. As such, an ultrasonication bath is not suitable for use in an integrated, automated assay system or for use by assay technicians that are not highly skilled. Disadvantageously, the ultrasonication bath of Sizto et al. cannot be incorporated into an assay device or assay system that is small, robust, inexpensive, easy to use. The ultrasonication bath would also not be suitable for a disposable device.
Many assay techniques detect the binding of molecules in solution to reagents located at a solid phase. The binding of molecules to reagents on a solid phase can be measured directly, for example, by surface plasmon resonance. Alternatively, by attaching a label to a molecule in solution, the binding of the molecule to a surface can be determined by measuring the amount of label located on the surface. Typical labels used in assays include enzymes, fluorescent molecules, radioactive isotopes, chemiluminescent molecules, electroactive molecules, and colloidal particles. For more description of the field, the reader is referred to Nonradioactive Labeling and Detection of Molecules, Kessler, C., ed., Springer-Verlag, Berlin 1992; The Immunoassay Handbook, Wild, D., ed., Stackton Press, New York 1994; and Keller, G. H.; Manak, M. M. DNA Probes, 2nd Ed., MacMillan Publishers Ltd., London, 1993.
One particularly useful detection technique is electrochemiluminescence (ECL). In ECL, electron transfer reactions at or near an electrode causes a label to adopt an electronically excited state. The excitation level of the label decays through emission of a photon which can be photometrically detected. Derivatives of ruthenium tris-bipyridyl (TAG1) are widely used as ECL labels. Further details regarding ECL detection techniques can be found in Bard et al. (U.S. Pat. No. 5,238,808) and Knight et al., 1994, Analyst, 119:879–890. While ECL monitoring of binding reactions in solution has been described, it is noted that a wide variety of ECL-based binding assays utilize binding reagents located on a solid-phase support. For example, the solid-phase support may consist of a magnetic bead that is deposited on an electrode surface (published PCT WO 92/14138 and Yang, H.; Leland, J.; Yost, D. Massey, R.; Bio/Technology 12 (1994) 193–194). Alternatively, an electrode (e.g., a fibril-polymer composite electrode) may be derivatized so as to provide a solid-phase support, for example, as described in copending U.S. application Ser. No. 08/932,110 filed on even date herewith, and PCT Application No. PCT/US97/16942 (WO 98/12539) filed on even date herewith, both of which are incorporated by reference above.